The ultimate goal of this project is to understand at the molecular level how the oxirane group of fosfomycin, the cyclohexene moiety of spinosyn A, and the annelated oligocyclobutane ring system of ladderane lipids are biosynthesized. Fosfomycin is a clinically useful antibiotic and chemically versatile synthon in the research and development of new therapeutics largely on account of its reactive epoxide ring. Spinosyn A is an environmentally green commercial insecticide with activity against insects detrimental to both human health and harvested plants. Ladderane lipids are a necessary component of the intracytoplasmic compartment of bacteria responsible for anaerobic ammonia oxidation (anammox). This process is employed in the bioremediation of nitrogen-contaminated wastewater. Therefore, the continued development of these compounds in the interest of public health requires a more complete understanding of the chemistry underlying their biosyntheses. To achieve these goals, detailed enzyme analysis will be coupled with chemical synthetic methods to develop mechanistic probes specific to the unique challenges each system presents. The mononuclear non-heme iron enzyme HppE, which is responsible for the oxirane of fosfomycin, will be investigated using a combination of 18O kinetic isotope effects, radical clock probes, designer substrate analogues, and spectroscopic methods. These techniques will be used to characterize the radical intermediates of HppE catalysis and to identify the reactive iron-oxygen species (FeIII-OO versus FeIV=O) responsible for their formation. The enzyme SpnF, which catalyzes the [4+2]-cycloaddition responsible for the construction of the cyclohexene ring of spinosyn A, will be investigated to verify whether it is indeed the first- confirmed natural Diels-Alderase. This hypothesis will be tested using secondary deuterium kinetic isotope effects and further characterized via thermodynamic and kinetic study of the cycloaddition reaction. The biosynthetic pathway of ladderanes is believed to involve polyunsaturated fatty acids with cyclization proceeding through B12-dependent radical SAM chemistry. Therefore, the putative biosynthetic gene cluster from Kuenenia stutgartiensis will be interrogated by reconstituting the ful pathway in vitro using chemo- enzymatically prepared substrates so as to characterize the key cyclization reactions. These studies will significantly enhance our understanding of the diverse chemistry and enzymology (non-heme iron-dependent oxidases, B12-dependent radical SAM enzymes, Diels-Alderases) of biological cyclization reactions, which can be exploited in future combinatorial biosynthetic endeavors to generate novel and structurally diverse compounds with therapeutic potential.